Process for the preparation of daunorubicin

ABSTRACT

The antibiotic daunorubicin is prepared by aerobically cultivating Streptomyces bifurcus, strain DS 23,219 (NRRL 3539), or a daunorubicin-producing mutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances, and separating daunorubicin formed during the culture.

United States Patent [191 Mancy et a1.

[4 1 Apr.1, 1975 PROCESS FOR THE PREPARATION OF DAUNORUBICIN [75] Inventors: Denise Mancy, Val-de-Marne; Jean Florent; Jean PreudHomme, both of Paris. all of France [30] Foreign Application Priority Data Nov. 18 1968 France 68.174206 [52] US. Cl 195/80 R [51] Int. Cl C12d 9/00 [58] Field of Search 195/80 [56] References Cited FOREIGN PATENTS OR APPLICATIONS 985,598 3/1965 United Kingdom 195/80 OTHER PUBLICATIONS Brazhnikova et al.. Physicochemical Properties of Antitumor Antibiotic Rubomycin Produced by Actinomyes Coeruleorubidus CA. Vol. 66, 1967, 7952lq.

DiMareo et al., Daunomycin. A New Antibiotic of the Rhodomycin Group," C.A. Vol 61, 1964. 12394c. DiMareo et al., Daunomycin, an Antibiotic for Tumor Treatment, C.A. Vol 62. 1965, l6922a. Tong et 211., Identity of Rubidomycin and Daunomycin." C.A. Vol.68, 1968. 332239.

Primary Eraminer-Joseph M. Golian Attorney, Agent, or Firm-Stevens, Davis, Miller & Mosher [57] ABSTRACT The antibiotic daunorubicin is prepared by aerobically cultivating Streptomyces bifiu'cus, strain DS 23,219 (NRRL 3539), or a daunorubicin-producing mutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances. and separating daunorubicin formed during the culture.

5 Claims, No Drawings PROCESS FOR THE PREPARATION OF DAUNORL'BICIN This invention relates to a new process for the preparation of the antibiotic daunorubicin. also known as 13,057 R.P. or rubidomycin.

In British Pat. No. 985.598 entitled "Improvements in or relating to Antibiotics and their preparation" ap' plied for on May 16. 1963 and granted to Rhone- Poulenc S.A.. the assignees of the present application. there is described the antibiotic designated 9.865 R.P.. its three principal constituents designated 13.213 R.P.. 13.057 R.P. (or daunorubicin) and 13.330 R.P.. the preparation of the antibiotic 9.865 R.P. by culture of Streptomyces 8.899" (NRRL 3046) or"Streptomyces 31.723" (NRRL 3045) in an appropriate medium. and the separation of the antibiotic 9.865 R.P. into its three constituents. I

The formula of daunorubicin is known. inter alia by the Belgian Pat. No. 724.531. as

ca ca 3 l c o l OH l n r -w CILO 0 0a O-Cll-CH cH-ca-cn-c1-r, 3 2 .l .1

The present invention relates to a new process for the preparation of daunorubicin by cultivation under aerobic conditions of a new microorganism more completely identified hereinafter. which belongs to the genus Streptomyces. The new microorganism, designated by the name Streptomyces bijiu'cus. strain DS Srrepimnycex hifilrcus. strain DS 23.219 forms cylindrical spores which measure 0.4 to 0.5 L./1.0 to 1.2 ,u.. 1ts sporiferous filaments, which are straight or slightly flexuous, 'are inserted onto the aerial filaments which support them individually or in clusters containing a reduced number of elements; the principal filament frequently gives rise in its end portion to only two elongated filaments, which gives the sporophore a rather peculiar forked appearance.

Generally. Streptomyces hifurcus. strain DS 23.219 develops a yellowish to light pink. brownish pink or reddish brown vegetative mycelium on synthetic nutrient media. and produces soluble pigments of an orange-pink to violet-pink colour. On organic media.

' its vegetative .mycellum is yellow-brown to orangebrown and the soluble pigments which it produces colour the agar orange-brown to reddish brown. lt'does not produce a melanin pigment on organic media and I does not produce H 5. It forms nitrites from nitrates both on organic media and on synthetic media. liquifies gelatine. slmvly.peptonises milk and utilises cellulose.

In Table 1 which follows there are given the cultural characteristics and biochemical properties of Streptomyces bifurcus. strain DS 23.219 on a certain number of nutrient agars and nutrient broths usually employed to examine the appearance of strains of Streptomyces. Unless otherwise stated. these characteristics and properties are thoseexhibited by cultures of about 2 to 3 weeks at C.. which have reached a good stage of de- 23.219". has been isolated from a soil sample taken inc the Val-de-Marne department of France. A specimen of this new microorganism has been deposited in the United States Department of Agriculture. Northern Regional Research Laboratory. at Peoria. 111. USA...

under the number NRRL 3539. Specimens of this microorganism may be obtained from this Laboratory on reference to the present patent application.

This new strain of Streptomyces was isolated by the following standard method: The sample of soil was suspended in sterile distilled water and the suspension diluted to different concentrations: small volumes of each dilution were spread over the surface of Petri dishes containing a suitable nutrient agar medium. After 15 days incubation at 26C., the colonies of microorganisms to be isolated were pricked out and transplanted onto agar slopes in order to obtain more abundant cultures.

For reasons given later, the strain Streptomyces DS 23.219 must be considered as a new species, designated by the name Streptomyces bifm-cus because of the fact that a rather peculiar form of sporophore is very frequently found in its cultures, which ends in a dichotomic division and thus presents two elongated branches attached to the end of a principal filament.

velopment. A certain number of the culture media employed were prepared in accordance with the formulae indicated in The Actinomycetes" (S.A. Waksman. Chronica Botanica Company. Waltham. Mass. U.S.A.. 1950, p.193-197); in this case. they are indicated by the letter W followed by the number given to them in The Actinomycetes". The references or compositions of the other culture media are as follows:

/ Ref. A Hickey and Tresner-sAgar" T. G. Pridham et al. 4 Antibiotics Annual. 1956-1957, p.950 Ref. B K. L. Jones Journal of Bacteriology. 5.7.

142. 1949 y Ref. C FormulaW-23 with the addition of 271 of agar Ref. D Yeast Extract Agar T. G. Pridham et al. Antibiotics Annual, 1956-1957, p.950 Ref. E. Tomato Paste Oatmeal Agar" T. G. Pridham et al. Antibiotics Annual, 1956-1957,

p.950 Ref. F. W. E. Grundy et al. Antibiotics and Chem. 2, 401. 1952 Reg. G Inorganic Salts Starch Agar T. G. Pridham et al. Antibiotics Annual, 1956-1957, p.951 Ref. -H corresponds to formula W-l, with 30 g. of

sucrose replaced by 15 g. of glucose Ref. 1 corresponds to formula W-l, with 30 g. of

sucrose replaced by 15 g. of glycerine Ref. .1 Plain gelatine" prepared in accordance with the instructions in Manual of Methods for Pure Culture Study of Bacteria Society of American Bacteriologists, Geneva, N.Y., 11 Ref. K Manual of Methods for Pure Culture Studyof Bacteria Society of American Bacteriologists, Geneva, N.Y., 11

Ref. M corresponds to formula W l of sucrose replaced by lS g. of glucose Ref. N corresponds to-formula W- l 8. with the sucrose omitted ,and replaced by small strips of filter paper pz trtiallyimm ersed in the liquid 8. with 30" g.

, 4 Ref. 0 Skimmed milk as aeornmer'cial powder. re-

constituted in accordance with the manufacturers instructions I Ref. P The Actinomycetes". vol. 2. p.333 No.

5 K 42 S. A. Waksmam The Williams and Wilkins Company. Baltimore. 1961 Ref. Q H. D. Tresner and F. Danga Journal of "Bacteriology, 76. 239-244. l958.

TA BLE l Culture Degree of Vegetative mycelium Aerial structure Soluble pigment Observations and medium development (\'.m.) or-underside (comprising the biochemical of the culture combination of the properties aerial mycelium and the sporulation) Hickey and v vGood V.m. light yellow Light grey. Very Very slightly Cylindrical spores Tresner agar brown. Well moderately pink brownish measuring 0.4 to (RefuA) developed; developed. (l.5;.t./l.0 to l.2;1..

' Straight or slihtly flexuous ilaments. Frequent sporophores of bifurcated shape. Bennett agar Fairly good V.m. yellowish to Nil Nil (Ref. B) light yellowish brown Emerson agar Fairly good V.m. yellowish to Nil Weak pink- (Ref. C) light yellowish brown brown Pridham yeast Good V.m. light orange- Light blue-grey. Light reddish extract agar brown Moderately orange-brown (Ref. D) developed. Pridham out Good V.m. light orange- Blue-grey. Orange-brown and tomato brown Fairly well agar (Ref. E) I deve oped. Glucosct Fairly good V.m. brownish orange Nil Slightly pink peptone agar orange-brown (W-6) Nutrient agar Very moderate V.m. light brownish Nil Nil (W-S) v vellow Krainsky Moderate V.m. colourless to Nil Light greyish Good solubilicalcium malate light brownish pink pink sation of the agar (Ref. F) calcium malate Ovalbumin agar Poor V.m. colourless to Ntl Nil 7 (W-lZ) light pink Glucosc- Fairly good V.m. light orange- Blue-grey. Very Light reddish asparagine brown to light redmoderately develorange. agar (W-Z) brown oped. Glycerine- Fairly good V.m. pink brown-yellow Pink-grey to blue- Orange-pink to asparagine to red grey. Very poorly red agar (W-3) developed. Starch-nit- Moderate V.m. reddish pink Light greyish. Light violet Hydrolysis of rate agar to light pink-brown Poorly developed. pink-brown the starch: (f-l0) I positive Pridham starch Fairly good -Unders1dc reddish Li ht bluish grey to Light reddish Cylindrical spores agar (Ref. G) orange-brown light pink-grey. brown measuring 0.4 to

Moderately devel- 0.5[L./1.0 to 12p. Fed. Straight or slightly flexuous sporiferous filaments. Frequent.- sporophores of bifurcated shape. Hydrolysis of the s r h: Czapek synthe- Good V.m. light yellowish Nil Vi i k m c tic agar with to reddish pink sucrose (W-l) I Czapek sy nthe- Good V.m. light yellowish Nil Pale pink to tie agar Wllh to light pink-brown Pale orange, glucose (Rcf.H) l Czapek sy nthe- Good V.m. violet-red Pink-grey to intense purpletic agar with violet-grey. violet. glycerine Moderately devel- Abundant (Rcf.l) oped. Culture on Good V.m. very well Nil Reddish brown P011110 developed, very thick I and very wrinkled. Light brownish to I g light reddish brown I IZI /r PUre Moderate V.r'n: whitish Nil Nil Liquefaction geat n (Rem) positive v The capacity of Streplomyces lnjiu'cus; strain DS 23,219. to utilise various sources of carbon andnitrogen to ensure its development hastbeen determined in accordance with the principle of the method of Pridham and Gottlieb (.l. of Bact. 56. l07l 14, 1948). The degree of development was observed on the base medium indicated by the authors, either replacing the glucose by the various sources of carbon respectively tested, or replacing the (NH SO by the various sources of nitrogen respectively tested. The results are indicated in the following Table.

TABLE 11 Sources of Sources of ,l carbon tested Utilisation nitrogen tested Utilisation D'Xylose NaNO .-l L-Arabinose NaNO -l: L-Rhamnose (NH SO D-Glucose (NH- ,HPO l'l D-Galactose Adenine D-Fructose Adenosine D-Mannose Uracil L-Sorbose Urea Lactose L-Asparagine Maltose Glycine -l" Sucrose Sarcosine Trehalose but slow DL-Alanine Cellobiose DL-Valine Raftinose DL-Aspartic acid Dextrin L-Glutamic acid 4- I lnulin -l- L-Arginine Starch L luysine Glycogen DL-Serin'e I Glycerine Dl-.-Threoni'ne Erythritol DL-Methionine Adonitol Taurine I .5 Dulcitol DL-Phen \5lalariine but slow D-Mannitol L-'l' \'ros ine D-Sorbitol DL-Proline +1 lnositol 'L Hydrt'txyproline -l Saliein Betaine: -l-' TABLE I Contii1ued; Culture Degree of Vegetative mycelium' Aerial structure Soluble pigment Observations and medium development (\'.m.) or underside comprising .the biochemical I ol the culture combination of the 3 properties aerial mycelium'and I :2

the sporulationl) i Nutrient broth Fairly good Yello'wish ring' Nil Nil Reduction of nicontalning trates to nitrinitrate (ReiIK) tes:- trongly I positive Dimmick Moderate Smallgreyish eol- Nil Nil Reduction of nitrates glucose-ni- I onies agglomer- I t to nitrites: trate broth ating on the surface positive (Ref. L) Czapek broth 1 Medium Pink \elums'thick. Nil Nil Reduction of nitrates with suerose t well developed to nitrites: (W-lX) positi\e' Czapek broth Medium Pink velum 'Nil Nil Reduction of nitrates with glucose to nitrites: (Ref. M) positiye Czapek broth Medium Small pink-white Pink-white Nil Reduction of nitrates with cellulose colonies on the to nitrites: I (Ref. N) paper protruding from positive-Utilisation the broth of the cellulose:

- positive Skimmed milk Good Yellowish ring Nil No coagulation (Ref. 0) Peptonisation slow.

1 only starting after one month's culture. pH'unehanged in v month. 5 Tyrosine- Moderate Light brownish pink Pink-white to Light brownish Formation of melyeast expink-grey. Mod- 7 pink: anin: negative tract agar erately developed. for the formation of melanin (Ref. P) I l Tresner and Good Light brownish yellow Nil Li ht brownish Production of Danga agar yefiow H- S: negative (R f- Q) I The combination of characteristicswhich SIrepI0m (es bifiircus, strain DS 23,2 19. presents does not allow it to be identified with any of the species already described. and for this reason it must be. considered as I representing a new species;

developing also a light reddish brown vegetative mycemyces fragilis. However, these three species show es-" however, it has no relationship tothe latter, which produces spiral sporophores and of which the sporulated .serial mycelium assumes a pink tint. whereas Slrepto Williams and Wilkins Company, Baltimore, 1961 page 158) there are described three other speciesshar- I ingwith Srreptomyces noiirsei the property of not forming a melanin pigment on organic media, of elaborating a reddish soluble pigment on potato and. of forming a pink to reddish vegetative mycelium on potato: Strepto myce's albogriseolus, SIfCPfU/PLVCES spiralis and Streptosential differencesin properties from the strain Strepto- I71 \C$"blftllill$', strain DS 23.3219, proving that they are indisputably species to which it cannot correspondz SHCPIOIHFd'S zilbogriseol is and Streplomyces spiralis form spiral sporophores, thereby placing them' in the section Spira-of Pridhamsclassification, whilst Sireptomyces bifurcus, strain DS 23,2l9, which only produces longs to the section Rectus-Flexibilis of this same clas-.

nitrogen iit'protein form. e.g. casein. lactalbumin. glusification. As regards Streplomyces fragilis. the pink colour which its aerial mycelium assumes whenit reaches sporulation shows that it is unrelated to Streplomyces 'HflUCUS. strain DS 23.219 of which the aerial mycelium. as already stated. assumes a light blue-grey colour when it is sporulated.

.S'n'epwmyces hzjiu'c'us. strain DS 23.2l9 can thus not be identified with any of the strains which come closest to it.

The new process for the preparation of daunorubicin according to the present invention comprises aerobically cultivating .S'Ireptomyces Influx-us. strain DS 23.219 (NRRL 3539), or a daunorubicin-producing mutant thereof. using an aqueous nutrient medium containing assimilable sources of carbon. nitrogen and inorganic substances; and separating the antibiotic daunorubicin formed during the culture.

The cultivation of SIrvpImnyc-ex lnfurcus. strain DS 23,2l9 by fermentation essentially produces daunoru bicin.but the other constituents 13213 R.P. and 13330 R.P. of the antibiotic 9865 R.P. are also produced. They are separated from daunorubicin during the course of the extraction and purification operations and may optionally be isolated. but the main object of the present invention is the preparation of daunorubicin.

The culture of Slreplmnyces bifurcus. strain DS 23,219 may be carried out by any of the known aerobic surface or submerged culture methods. the latter being preferred because they are more convenient. Conventional types of apparatus currently used in the fermentation" industry may be employed.

ln partlc'ular, the following sequence of operations may be adopted:

I yield of antibiotic is obtained after 2 ,ten and their hydrolysates, soyabean flour, peanut meal. fish mealfmeat extract, yeast extract, distillers solubles and corn steep liquor.

Of the inorganic substances added, some may have a buffering (Sr-neutralising effect. such as the alkali metal phosphates or alkaline earth metal phosphates, or the carbonates of calcium and magnesium. Others contribute to the ionic equilibrium needed for the development of S treplomyces blfurc'us, strain DS 23,219 and for the production of the antibiotic; examples of these are the chlorides and sulphates of the alkali metals and alkaline earth metals. Finally, some of them act more especially as activators of the metabolism of Streptomyces DS 23,219: to these belong the salts of zinc, cobalt, iron, copper and manganese.

The pH of the fermentation medium at the beginning of the culture should be within the range of 6.0 to'7.8, and preferably from 6.5 to 7.5. The optimum fermentation temperature is 30C., but satisfactory production is achieved at temperatures of from 23 to 33C. The rate of aeration of the fermentation vessels may vary between quite wide limits, but is has been found that an aeration rate of 0.3 to 3 litres of air per litre of broth per minute is particularly suitable. The maximum to 8 days of growth. but the period depends predominantly on the medium used.

From the foregoing it will be realised that the general conditions for the culture Streptomyces blfm'c'us, strain DS 23,219 for the production of daunorubicin may be widely varied and adapted as appropriate to the circumstances.

Daunorubicin may'be isolated from the fermentation broths by various methods. The culture broth can be filtered at a pH between 1.5 and 9 and, under these stregtogz ces biiurcus, strain D'S 25,212 stock .L culture on agar t culture in an agitated flask 'ino'culum culture in a fermentation vessel production culture in a fermentation vessel The fermentation medium must contain assimilable sources ofcarbon. nitrogen and inorganic substances.

and optionally growth-promoting factors; all these ingredients may be supplied as well-defined products or complex mixtures. such as are found in natural biological products of various origins.

Asv the assimilable source of carbon there may be used carbohydrates such as glucose, maltose, dext rins,

starch or other carbon-. hydrogenand oxygencontaining substances such as the sugar alcohols, e.g. mannitol, or certain organic acids. e.g. lactic or citric acid. Certain animal or vegetable oils such aslard ,oil or soyabean oil may be advantageously used instead of,

or in admixture with,carbon-, hydrogenand oxygencontaining substances.

A very wide range of suitable sources of assimilable nitrogenis available. The sources may be very simple chemical compounds such as nitrates, inorganic and,

organic ammonium salts, urea, or amino-acids. They may also be complex substances containing principally conditions, the major part of the active material passes into the filtrate. After washing with water, the filter cake retains practically no active material. it is advantageous to carry out this operation in acid medium, and particularly one acidified'with oxalic acid to a pH between 15 and 2. It is also possible to carry out the filtration at a pH between 2 and 7, preferably near to 2, in the presence of an aliphatic alcohol containing from 1 to 3 carbon atoms.

In these extraction operations, daunorubicin is obtained in aqueous or aqueous-alcoholic solution, and it is then brought intoorganic solution by extraction .with a water-immiscible organic solvent such as butanol, methyl isobutyl ketone,-ethyl acetate or chloroform, at a pH between 5.5 and 9 preferably about 7.5. This extraction is optionally preceded by a treatment on an ion-exchange resin, in which case the aqueous solution is adjusted to a pH of about 4 and then treated with a cation exchange resin. Daunorubicin is eluted, preferably with methanol containing 10% of water and 1% of sodium chloride. The eluate is then concentrated to remove the alcohol and extracted as described above.

The fermentation broth may also be directly .extracted with a water-immiscible organic solvent such as butanol. ethyl acetate or chloroform, at a pH between 5.5 and 9, preferably about 7.5. In this case. all the ac tive material passes into the organic phase. which is separated from the aqueous phase by the usual methods.

Whatever the method of extraction chosen. daunorubicin is finally obtained in organic solution. It

may be advantageous at this stage to purify the antibi-v otic by successively bringing it into aqueous solution and then into organic solution by varying the pH. The crude antibiotic may be isolated from the organic solution last obtained by concentration or precipitation with a poor solvent for the antibiotic such-as hexane.

To obtain daunorubicin in a purer state. all the usual methods may be employed. such as chromatography on various adsorbent substances. counter-current distribution or partition between various solvents.

Daunorubicin may also be converted to acid addition salts by treatment with acids, for example hydrochloric acid. Such salts may be purified by application of conventional methods.

Daunorubicin obtained by this new method and its acid addition salts exhibits characteristics identical to those of the antibiotic 13057 R.P.. and of its acid addition salts, described in British Patent No. 985,598.

The following Examples illustrate the invention.

EXAMPLE 1 Peptone (1200 g.). meat extract (600 g.), Cerelose (1200 g.), agar (240 g.) and tap water (sufficient to make up to 110 litres) are introduced into a 170-litre fermentation vessel.

After the pH of the mixture has been adjusted to 7.20 with lON sodium hydroxide solution (120 cc.), the medium is sterilised by bubbling steam at 122C. through it for 40 minutes. After cooling, the volume of the broth is 120 litres and the pH is 6.65. The broth is then inoculated with a culture (200 cc.) in an agitated Erlenmeyer fiask of Strepmmyr'es hiflll'tllS, strain DS 23,219 (NRRL 3539). The culture is developed at 27C. for 30 hours with agitation and aeration with sterile air; it is then ready for inoculation of the production culture.

'The production culture is carried out in an 800-litre fermentation vessel charged with distillers solubles (4 kg), shelled beans 12 kg. soya bean oil (8 litres). sodium chloride (2 kg.), cobalt chloride. 6 H (8 g.), and tap water (sufficient to make up to 330 litres). After adjustment of the pH of the mixture to 7.70 with 10N sodium hydroxide solution (400 cc.), the medium us sterilised by bubbling steam at 122C. through it for 40 minutes. After cooling, the volume of the broth is 360 litres. 1t is made up to 400 litres by addition of a sterile aqueous solution (40 litres containing Cerelose (6 kg). The pH is then 6.80.

The broth is then inoculated with 40 litres of the inoculum culture described above from the 170-litre fermentation vessel. The production culture is carried out at 27C. for 170 hours with agitation, using a motor revolving at 260 revolutions per minute, and aeration with a flow of 30 m/hr. of sterile air. The pH of the culture medium is then 7.30 and the volume of the broth is 305 litres. The quantity of daunorubicin (13,057 R.P.) present in the medium is 22.5 ug./cc.

1 0 EXAMPLE 2 200 Litres of the culture broth obtained from the fermentation described in Example 1 are placed in a vat equipped with 'a stirrer and a steam heating coil. Oxalic acid (6 kg.) is added and the mass is stirred and heated to 50C. The stirring and the said temperature are maintained for 1 hour 30 minutes, after which time a filtration adjuvant;(30 kg.) is'added and then the suspension is filteredon a filter-press. The filter cake is washed on the filter with water (100 litres). The filtrate, the volume of which is 260 litres. is cooled to +15C. and the pH is adjusted to 4.5 by addition of 10% sodium hydroxide solution.

The filtrate is passed through a column containing Amberlite lRC-50 (6 litres) in acid form so as to pass through the resin bed from the top downwards at a flow rate of 15 litres/hour. The column is then washed with water (30 litres) circulating in the same direction as the filtrate and at the same flow rate. followed by methanol containing 50% of water (30 litres) at a flow rate of 15 litres/hour circulating from the bottom upwards and then with methanolcontaining 10% off water (50 litres) again at the same flow rate and from the bottom upwards.

The effluent and the washings are discarded. and the column is eluted with a solution circulating from the top downwards through the resin, the solution consisting of sodium chloride (10 g.), water (100 cc.) and methanol (sufficient to make up to 1000 cc.). The eluate is collected as soon as an orange-red colouration appears and until this colour disapppears. A volume of 50 litres is thus obtained. which contains the major part of the antibiotic. The eluate is concentrated under reduced pressure (2 mm.Hg) at 35C. to a volume of 10 litres.

The concentrate is successively extracted at pH 8.5 with 3 amounts of chloroform (5 litres each). The chloroformic extract is concentrated at 30C. under reduced pressure (5 mm.Hg) to a volume of 50 cc. The antibiotic is precipitated in the form of the base by addition of hexane (500 cc.). and is then filtered off washed and dried to give finally a crude base (4.9 g.) containing 64% of daunorubicin.

EXAMPLE 3 The product (6.8 g.), obtained as described in Example 2 containing 64% of daunorubicin. is suspended in water (40 cc.) and dissolved by progressively adding 1N hydrochloric acid.(7 cc.). The solution is clarified by filtration and daunorubicin hydrochloride crystallised by the slow addition of acetone 1 litre). The crystals thus obtained are filtered off, washed with acetone (50 cc.) and dried at 50C. under reduced pressure (5 mm.Hg) for 15 hours. A crystalline hydrochloride (3.3 g. titrating of daunorubicin, is obtained in a yield EXAMPLE 4 The product obtained as described in Example 3 (3 g.) is. dissolved in methanol (10 cc.) and chloroform cc.) is then added progressively, causing the daunorubicin hydrochloride to recrystallise. The crystals thus obtained are filtered off, washed with chloroform (20 cc.) and dried at 50C. under reduced pressure (5 mm.Hg) for 15 hours to give the recrystallised hydrochloride (1.8 g.) containing 90% of daunorubicin. Treatment of the crystallisation mother liquors with hexane 200 cc.) caused the precipitation of a second quantity of daunorubicin. After filtratiomwashing with hexane (50 cc.) and drying at 50C. under reduced pressure (5 mm.Hg. daunorubicin (l g.). which can be recycled under the conditions of Example 3. is obtained.

EXAMPLE 5 Daunorubicin hydrochloride (2.4 g. obtained as described in Example 4. is suspended in butanol (40 cc. After half an hour's stirring. the crystals are filtered off. washed with butanol cc.) and dried at 60C. under reduced pressure (0.5 mm.Hg) for 8 hours. Pure daunorubicin hydrochloride (2.1 g.) is obtained in a yield of 88%.

We claim: 1

1. Process for the production of daunorubicin which comprises aerobically cultivating SII(])I()HI bifur- LII 12 cus, strain DS 23.219 (NRRL 3539). or a daunorubicin-producing mutant thereof, using an aqueous nutrient medium containing assimilable sources of carbon. nitrogen and inorganic substances, and separatin daunorubicin formed during the culture.

'2. Process according to claim 1 in which the culture is effected under submerged aerobic culture conditions commencing at a pH withintherange, of 6.0 to 7.8 at a temperature of from 23 to 339C.

3. Process according to claim 2 in which the pH of the nutrient medium at the beginning of the culture is from 6.5 to 7.5.

4. Process according to claim 2 in which the temperature of the culture is 25 to 30C. v v

5. Process according to claim 2 in which the culture medium is aerated at a rate of from 0.3 to 3 litres of air per litre of medium per minute. 

1. PROCESS FOR PRODUCTION OF DAUNORUBICIN WHICH COMPRISES AEROBICALLY CULTIVATING STREPTOMYCES BIFURCUS, STRAIN DS 23,219 (NRRL 3539), OR A DAUNORUBICIN-PRODUCING MUTANT THEREOF, USING AN AQUEOUS NUTROGEN AND INORGANIC SUBSTANCES, LABLE SOURCES OF CARBON, NITROGEN AND INORGANIC SUBSTANCES, AND SEPARATING DAUNORUBICIN FORMED DURING THE CULTURE.
 2. Process according to claim 1 in which the culture is effected under submerged aerobic culture conditions commencing at a pH within the range of 6.0 to 7.8 at a temperature of from 23* to 33*C.
 3. Process according to claim 2 in which the pH of the nutrient medium at the beginning of the culture is from 6.5 to 7.5.
 4. Process according to claim 2 in which the temperature of the culture is 25* to 30*C.
 5. Process according to claim 2 in which the culture medium is aerated at a rate of from 0.3 to 3 litres of air per litre of medium per minute. 